Burkholderia pseudomallei is a bioterrorist threat. With the best current therapies, lethality is typically as high as 40%. The overall goal of this proposal is the development of new drugs against this organism. In Phase I, the high sequence similarity between B. pseudomallei and its less virulent relative Pseudomonas aeruginosa was exploited to build innovative screens for rapid, safe discovery of effective therapeutic agents. Three key discoveries of the Phase I research established the feasibility of this approach: (a) cross-species complementation is efficient enough between these two species to permit construction of P. aeruginosa deletions surviving by virtue of expression of the 6. pseudomallei ortholog in almost 50% of cases attempted; (b) lac regulation is adequate to deplete targets to the point of limiting cell growth in about 33% of cases tested; (c) expression profiling by microarray hybridization revealed genes specifically up-regulated for 4 of 5 depleted target strains tested. Promoters from target depletion-responsive genes were fused to the P. luminescens luxCDABE operon to generate luciferase reporter assays in P. aeruginosa for inhibition of B. pseudomallei targets. Construction and implementation of a model whole cell luciferase reporter screen for inhibitors of GyrA established the feasibility of such screens for antibacterial drug discovery. In Phase II, three new reporter assays for inhibitors of validated B. pseudomallei targets will be used to screen two diverse chemical libraries representing over 150,000 compounds. Confirmed hits will be subjected to a panel of secondary assays for validation as target specific, potent on B. pseudomallei cells, and non- cytotoxic to mammalian cells in culture. Validated hits will be prioritized by potency and drug-like features and optimized by rational drug design aided by structural information for several inhibitor-target co-crystals. Lead compounds will be tested in mice to determine the maximum tolerated dose, pharmacokinetic properties, and efficacy in a B. pseudomallei infection mouse model. In addition, utilizing the expertise gained in Phase I studies, P. aeruginosa whole cell reporter assays will be developed and screened for inhibitors of at least three additional B. pseudomallei targets. In Phase III, a potent, safe, orally active B. pseudomallei inhibitor will be advanced into IND enabling toxicology and safety pharmacology studies and file an IND. [unreadable] [unreadable] [unreadable]